JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

 JoVE Biology

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

1, 1, 1

1Yale Stem Cell Center, Department of Genetics, Yale School of Medicine

Article
    Downloads Comments Metrics

    You must be subscribed to JoVE to access this content.

    This article is a part of   JoVE Biology. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.

    Recommend JoVE to Your Librarian

    Current Access Through Your IP Address

    You do not have access to any JoVE content through your current IP address.

    IP: 54.226.151.20, User IP: 54.226.151.20, User IP Hex: 920819476

    Current Access Through Your Registered Email Address

    You aren't signed into JoVE. If your institution subscribes to JoVE, please or create an account with your institutional email address to access this content.

     

    Summary

    A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

    Date Published: 4/03/2012, Issue 62; doi: 10.3791/3804

    Cite this Article

    Kim, K. Y., Hysolli, E., Park, I. H. Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP. J. Vis. Exp. (62), e3804, doi:10.3791/3804 (2012).

    Abstract

    Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

    Protocol

    1. Reprogramming by Retrovirus Expressing Reprogramming Factors

    1. Human fibroblasts are cultured in fibroblast medium (10% FBS in DMEM with Pen/Strep).
    2. One day before infection, plate 1x105 human fibroblasts into one well of a 6-well plate.
    3. Aspirate medium to remove dead cells and add 2 ml of fresh fibroblast medium. Add protamine sulfate at a final concentration of 5 μg/ml.
    4. Carefully add the appropriate amount of each GFP-expressing virus corresponding to a multiplicity of infection (MOI) 55.
    5. One day after infection, remove the viral supernatant, wash three times with 2 ml PBS, then add 2 ml fibroblast medium.
    6. Three days after infection, check the GFP fluorescence and replenish the well with 2 ml fibroblast medium.
    7. Four days after infection, plate 1x104 /cm2 of irradiated mouse embryonic fibroblast (MEFs) feeder cells in fibroblast medium onto a 10-cm Petri dish coated with 0.1% gelatin. Incubate at 37 °C overnight.
    8. Five days after infection, detach infected human fibroblasts with 1 ml 0.05% typsin/EDTA for 5 minutes at 37 °C, and centrifuge for 5 minutes at 200 g. Aspirate the medium and resuspend the cells with 10ml of fibroblast medium. Transfer the cells into a pre-coated 10-cm plate.
    9. After 24 hours, replace the medium with hESC culture medium (20% Knock-Out serum replacement, DMEM/F12, 0.1 ml Non-essential Amino Acids, 4 ng/ml bFGF, Pen/Strep/Glutamate, beta-merceptoethanol). Change the medium daily. ESC-like colonies will start to appear by day 20-27 after infection.

    2. Isolation and Expansion of iPSCs

    1. Under a fluorescence microscope, check for the absence of GFP fluorescence in a colony that shows a similar morphology to hESCs.
    2. Using a 10 μl pipette, pick individual iPSC colonies and place them into one well of a gelatin- and MEF-coated 12-well plate and supplemented with hESC medium. Change medium daily.
    3. For passaging, wash the plate with 1 ml of DMEM/F12, then add 0.5 ml of collagenase, and incubate for 10 minutes at 37 °C.
    4. Wash the cells twice with DMEM/F12.
    5. Add 2 ml of fresh hESC medium. Using a cell lifter, break up the colonies into small pieces and detach remaining cells from the plate.
    6. Transfer the resuspended pieces of colonies into one well of a gelatin- and MEF- coated 6-well plate.

    3. Immunofluorescence Analysis of Pluripotent Markers

    1. Wash the cells three times with PBS and fix with 4% paraformaldehyde for 20 min at room temperature.
    2. Gently wash the cells three times with PBS and permeabilize with 0.2% Triton X-100 in PBS for 30 min.
    3. Block non-specific binding by incubating cells with 3% BSA in PBS for two hours.
    4. Incubate the cells with primary antibody overnight at 4 °C.
    5. Wash the cells three times with PBS and incubate the cells with specific secondary antibody for one hour at room temperature, shielding from light.
    6. Wash the cells three times with PBS and add DAPI during the last wash followed by incubation at room temperature for 5 minutes.
    7. Detect the staining with a fluorescence microscope.

    4. Quantitative Real-time PCR Assay for the Pluripotent Markers

    1. Isolate total RNA from the human iPSCs derived from human fibroblasts using Qiagen's RNeasy kit.
    2. Synthesize the first-strand cDNA using SuperScript II Reverse Transcriptase.
    3. Perform qPCR to detect pluripotency genes using primers previously reported.6

    5. Representative Results

    1. Morphological change during reprogramming
      We infected human fibroblasts BJ1 and Detroit 551 with a cocktail of retroviruses carrying OCT4, SOX2, KLF4 and MYC, and were able to detect morphological changes during reprogramming (Figure 1). Twenty-one days after infection, we recognize small iPSC colonies by their hESC-like morphology. Furthermore, we recognize iPSCs by the GFP fluorescence. Pluripotent stem cells, such as ESCs and iPSCs, express the molecular machinery to repress the proviral gene expression7-9. Our unique retroviral vector expresses GFP together with reprogramming genes by retroviral LTR. Thus, cells continuously expressing GFP are considered to express transgenes without proviral gene silencing Faithfully reprogrammed iPSC colonies that acquire the pluripotency molecular network show the absence of GFP expression (Figure 2)10.
    2. Characterization of pluripotency of human iPSCs
      We analyzed colonies derived from Detroit-551 fibroblasts via immunohistochemistry with Tra-1-81, Tra-1-60, SSEA-4, SSEA-3, OCT4 and NANOG antibodies (Table 2). Successfully reprogrammed iPSCs express all of these markers (Figure 3A). We also analyzed gene expression via quantitative RT-PCR analysis. We observed that the expression of OCT4, SOX2, KLF4, MYC and NANOG was significantly increased compared to the parental fibroblast cells but commensurate with that of H9 hESCs (Figure 3B).

    Figure 1
    Figure 1. Morphological changes of retrovirus-infected human fibroblasts. (A-D) Progressive morphological change in colonies from Detroit-551 fibroblasts infected with reprogramming factors. Day 5 (A), Day 10 (B), Day 14 (C), day 21 (D). Cells show the hESC-like morphology after 21 days.

    Figure 2
    Figure 2. Representative GFP fluorescent expression in cells undergoing reprogramming. BJ and Detroit 551 fibroblasts were infected with retrovirus expressing four reprogramming factors4, and incubated in hESC medium for four weeks. BJ fibroblasts (A, B) and Detroit 551 (C, D) show similar morphological. From day 21, GFP negative colonies start to form, which represent the bona fide iPSCs10. (E, F) show transformed Detroit 551 cells that have not undergone proper reprogramming. (A, C, E) colonies under phase contrast view. (B, D) properly reprogrammed cells that show the GFP silencing. (F) bright GFP expression from transformed colony.

    Figure 3
    Figure 3. Characterization of the human induced pluripotent stem cells. (A) Human 551-iPS-K1 cell colonies express markers common to pluripotent cells. DAPI staining indicates the total cell content per field. (B) Quantitative real time-PCR (RT-qPCR) for expression of OCT4, SOX2, KLF4, MYC in parental fibroblast, 551-iPS-K1 iPSCs, and H9 human embryonic stem cells (hESCs). Data were normalized against β-actin housekeeping gene and plotted relative to the expression level in the parental fibroblast cells4.

    Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    Expression of four transcription factors reprograms human fibroblasts to iPSCs. Many attempts were made to generate human iPSCs using non-integrating or non-genetic approaches to generate clinically safe iPSCs. So far, these methods show extremely low efficiency and require further optimization to improve the reproducibility11-14. Retro- or lentiviral methods are readily used to derive and apply iPSCs for human in vitro disease models, which are less dependent on the safety issues caused by viral integration. Reprogramming method described here is available for the efficient derivation of human iPSCs. Selection of human iPSCs is primarily based on colony morphology that resembles human ESCs. More importantly, our method takes advantage of the feature of silencing of the retroviral long terminal repeats (LTR) in pluripotent stem cells7,15. The retroviral vector used in this protocol contains the GFP gene linked to the reprogramming factors via an Internal Ribosome Entry Sequence (IRES) and5. GFP expression is driven by proviral LTR. The fibroblasts infected with these viruses initially show the bright GFP expression. Once fully reprogrammed, iPSCs will lose GFP expression, which is easily visualized under a fluorescence microscope. In this protocol, we described the generation of iPSCs from fetal and neonatal fibroblasts (Detroit 551 and BJ1). However, this retroviral vector has been used to generate iPSCs from normal adult fibroblasts as well as a variety of patients with Mendelian and complex disorders4,16,17.

    Previously we have analyzed the change in cellular surface markers during human somatic cell reprogramming10. There is a progressive change in cell surface markers. Fibroblasts express CD13, which is repressed by the expression of reprogramming. Cells undergoing reprogramming start to express SSEA4 together with GFP. Then, they lose the expression of GFP via proviral silecing and express additional pluripotency maker TRA1-6010. The expression of TRA1-60 is well correlated with GFP silencing and the well-developed teratoma formation, suggesting that TRA1-60 is a marker for faithfully reprogrammed iPSCs. GFP silencing is the alternative marker for TRA1-60 expression and allows the identification of iPSC colonies without laborious live cell staining. Using the loss of GFP expression as a marker for iPSCs, stem cell scientists who have no prior experience in reprogramming will readily and consistently isolate iPSCs.

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    We have nothing to disclose.

    Acknowledgements

    This work was funded by Yale School of Medicine and Child Health Research Award from Charles Hood Foundation.

    Materials

    Name Company Catalog Number Comments
    DMEM/F12 Invitrogen 11330057 80%
    Knockout Serum Replacer Invitrogen 10828-028 20%
    L-Glutamine (200 mM) Invitrogen 25030081 2 mM
    Nonessential Amino Acids (10 mM) Invitrogen 11140050 0.1 mM
    β-Mercapt–thanol (14.3 M) or MTG Invitrogen M-6250 0.1 mM
    bFGF-2 10 μg/ml GIBCO, by Life Technologies GF003AF 4 ng/ml
    Penicillin/Streptomycin EMD Millipore 15140-122 1%
    DMEM Invitrogen 11965118 90%
    FBS Invitrogen 10407028 10%
    Penicillin/Streptomycin EMD Millipore 15140-122 1%
    Table 1. Culture Medium
    OCT4 Abcam Ab19857 1:500
    SSEA3 EMD Millipore MAB4303 1:100
    SSEA4 BD Biosciences BD560218 1:100
    Tra-1-81 BD Biosciences BD560173 1:100
    Tra-1-60 BD Biosciences BD560174 1:100
    NANOG Abcam Ab21624 1:500
    Alexa-Flur 488 Invitrogen A11008 1:1000
    Alexa-Flur 555 Invitrogen A21422 1:1000
    DAPI Invitrogen D1306 1:5000
    pMIG-OCT4 Addgene 17225
    pMIG-SOX2 Addgene 17226
    pMIG-KLF4 Addgene 17227
    pMIG-MYC Addgene 18119
    Collagenase type IV Invitrogen 17104019 1mg/ml
    Gelatin, Porcine Sigma-Aldrich G 1890 0.1%
    Triton Sigma-Aldrich X100-500ML 0.2%
    Paraformaldehyde Sigma-Aldrich 47608 4%
    BSA American Bioanalytical AB01800 3%
    MEF feeder cells EMD Millipore PMEF-N
    Cell Lifter Corning 3008
    Fluorescent microscopy: inverted microscope with GFP filter
    Table 2. Reagents and equipment

    References

    1. Murry, C.E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 132, 661-680 (2008).
    2. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131, 861-872 (2007).
    3. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 318, 1917-1920 (2007).
    4. Park, I.H., Zhao, R., West, J.A., Yabuuchi, A., Huo, H., Ince, T.A., Lerou, P.H., Lensch, M.W., & Daley, G.Q. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 451, 141-146 (2008).
    5. Park, I.H., Lerou, P.H., Zhao, R., Huo, H., & Daley, G.Q. Generation of human-induced pluripotent stem cells. Nature Protocols. 3, 1180-1186 (2008).
    6. Park, I.H., Zhao, R., West, J.A., Yabuuchi, A., Huo, H., Ince, T.A., Lerou, P.H., Lensch, M.W., & Daley, G.Q. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 451, 141-146 (2008).
    7. Hotta, A. & Ellis, J. Retroviral vector silencing during iPS cell induction: an epigenetic beacon that signals distinct pluripotent states. Journal of Cellular Biochemistry. 105, 940-948 (2008).
    8. Matsui, T., Leung, D., Miyashita, H., Maksakova, I.A., Miyachi, H., Kimura, H., Tachibana, M., Lorincz, M.C., & Shinkai, Y. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature. 464, 927-931 (2010).
    9. Wolf, D., & Goff, S.P. Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature. 458, 1201-1204 (2009).
    10. Chan, E.M., Ratanasirintrawoot, S., Park, I.H., Manos, P.D., Loh, Y.H., Huo, H., Miller, J.D., Hartung, O., Rho, J., Ince, T.A., et al. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27, 1033-1037 (2009).
    11. Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, II, & Thomson, J.A. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 324, 797-801 (2009).
    12. Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S., Ko, S., Yang, E., Cha, K.Y., Lanza, R., et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 4, 472-476 (2009).
    13. Warren, L., Manos, P.D., Ahfeldt, T., Loh, Y.H., Li, H., Lau, F., Ebina, W., Mandal, P.K., Smith, Z.D., Meissner, A., et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 7, 618-630 (2010).
    14. Ban, H., Nishishita, N., Fusaki, N., Tabata, T., Saeki, K., Shikamura, M., Takada, N., Inoue, M., Hasegawa, M., Kawamata, S., et al. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proceedings of the National Academy of Sciences of the United States of America. 108, 14234-14239 (2011).
    15. Wolf, D. & Goff, S.P. TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell. 131, 46-57 (2007).
    16. Park, I.H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M.W., Cowan, C., Hochedlinger, K., & Daley, G.Q. Disease-specific induced pluripotent stem cells. Cell. 134, 877-886 (2008).
    17. Kim, K.Y., Hysolli, E., & Park, I.H. Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome. Proceedings of the National Academy of Sciences of the United States of America. 108, 14169-14174 (2011).

    Comments

    0 Comments

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter